This application focuses on DNA-protein interactions that precisely time new rounds of chromosome replication, with the long-term objective of dissecting molecular mechanisms controlling bacterial growth. E. coli chromosomal DNA replication is triggered during each cell cycle by pre-replication complexes (pre-RC) that unwind origin (oriC) DNA. In normal pre-RC assembly, initiator DnaA interacts with at least eight binding sites at the correct cell cycle times. Our major goal is to understand how intrinsic nucleotide sequence of oriC determines temporal regulation of ordered assembly, the functional requirement for ATP-DnaA, synchronous firing of multiple origins, and one initiation per cycle control. Key experiments focus on newly discovered DnaA binding sites (I2 and I3), which are required for ATP-DnaA dependent unwinding, play a role in Fis and IHF regulation of pre-RC assembly, and contain the deoxyadenosine methyltransferase recognition sequence, GATC. The Specific Aims are as follows: 1. To use mutagenesis, DNA footprinting, and unwinding assays to test the hypotheses that DnaA binding sites are critically positioned in oriC and ordered DnaA binding is required for correct pre-RC assembly; 2. To evaluate ordered DnaA loading and the interplay between DnaA, Fis and IHF binding at oriC sites as determinants of initiation timing and synchrony using age-selected E. coli and flow cytometry; 3. To use mutagenesis, DNA footprinting and chemical assays to test the hypothesis that DnaA recognition sites 12, 13 and the IHF binding site are responsive to DNA methylation state and/or sequestration; and 4. To test the hypothesis, using mutagenesis and chemical assays, that DnaA binding sites within the A-T rich, 13-mer unwinding region regulate the location of strand separation and repress unwinding at high initiator levels. Our continued dissection of the triggering mechanism for chromosomal DNA synthesis in E. coli should provide new insight into the function of growth regulatory machinery in all living cells, particularly cell cycle-specific switches. Identification of new features of the pre-RC is also immensely important for understanding the control of bacterial growth, as well as cell growth defects, and can help to identify new targets used to guide the design of novel cell growth inhibitors.